Analysis of an estimated 5000 genetic disorders linked to hereditary disease and identification of ˜1.4 million single nucleotide polymorphisms (SNP) rely on gene amplification by PCR. Mutations in the β-globin gene, for example, result in sickle cell anemia and β-thalassaemia, with symptoms ranging from mild to severe anemia (Talmaci et al., 2004). Cancer, diabetes, hemophilia, cystic fibrosis, heart disease, musculoskeletal disorders and many other diseases can have a genetic basis. Increasing the speed and sensitivity of PCR could improve diagnosis of genetic diseases and enhance the development of therapeutic or prophylactic countermeasures.
SPR Detection of DNA: Label-free biomolecular interaction analysis (BIA) of DNA (Feriotto & Gambari, 2004; Goodrich et al., 2004) including PCR products (Kaie et al., 1999) has been evaluated in planar SPR sensors like commercially-available BIACore™ (BIACore) or Spreeta™ (Texas Instruments) analyzers. Single-base pair mismatches at or near the 3′ end of template/primer dsDNA could be distinguished by SPR based on relative binding efficiencies to DNA polymerase I (Tsoi et al., 2000). Sequencing of polynucleotides by interaction with immobilized DNA polymerase and SPR-based detection of ensuing effects has been suggested (Densham, 2002). These studies show DNA detection by SPR can reach 1-500 femtomolar levels (Goodrich et al., 2004; Park et al., 2002) with point mutation selectivity factors of ˜105:1 (Park et al., 2002), although simultaneous laser-induced amplification and real-time detection of template DNA have not been reported.
Extinction properties of plasmon resonant nanoparticles (NP) have been examined (Oldenburg et al., 1999; Westcott et al., 2001) and have been applied to detect bound DNA (Park et al., 2002; Schultz, 2003; Storhoffet al., 2002; Taton et al., 2000). These reports have not examined solid-phase amplification by polymerase or primers bound to planar- or nanoparticle-Au surfaces, but they show how ionic strength and chain length affect Au-oligonucleotide interactions including aggregation, binding, hybridization and denaturation.
Immobilizing Primers and Probes on Au Surfaces: Minisequencing on oligonucleotide microarrays is promising for large-scale DNA analysis and detection of single nucleotide polymorphisms (SNP) (Lindroos et al., 2001; Pastinen et al., 1997). Real-time SPR-induced minisequencing using primers or probes on Au surfaces has not been reported, although DNA has been immobilized on NP and hybridized NP-DNA conjugates have been detected by SPR. Storhoff et al. (2002) showed linking alkanethiol-capped poly-T, C or A to Au NPs prevented aggregation in ≦1M NaCl using 8- and 12-np oligos, and in 0.07-1.2M MgCl2 using larger oligos in the range 5- to 15-bp. Cleaving 5′ disulfide bonds of alkanethiol-capped oligonucleotides permitted Au-thiol covalent bond formation and gave surface coverages of ˜1013 strands/cm2 (Jin et al., 2003). This yielded ˜90, 160 and 250 single strands per 13, 31 and 50-nm NP, respectively. Alkanethiol-capped poly-T gave higher Au surface coverages than poly-C or -A, preventing non-specific Au-DNA interactions from amine or carbonyl functional groups and backbone phosphate groups and providing greater stability (Demers et al., 2002). Cyclic dithiane-epiandrosterone disulfide linkers and trithiol-capped oligonucleotides increased stability of DNA-oligo covalent bonds in the presence of dithiothreitol, which is present as a stabilizer in some PCR buffers (Shultz, 2003).
Probe Hybridization, Primer Annealing and Denaturation on Au Surfaces: Au-NP-immobilized alkanethiol-capped oligos that were hybridized to complementary oligos exhibited sharp melting profiles and higher melt temperatures (Tm) than analogous oligos with molecular fluorophore probes (Storhoff et al., 2002). This effect arose from multiple links between NP and local ionic strength effects. Melt-curve sharpness and Tm increased with immobilized probe density, with poly-A spacer length from 0 to 30 bp, and with NP size from 13 to 50 nm at constant concentrations of NP and target (Jin et al., 2003). Melting temperatures increased from 41 to 61.5° C. and hybridization rates increased as NaCl content increased from 0.05 to 1.0 M. Hybridization with immobilized oligos was not detected for NaCl<0.05 M at room temperature. Changing NaCl content ˜0.1M induced melting or denaturation at T<Tm.
Immobilizing Polymerase on Au Surfaces: Research on chromatin loops suggests template slides past DNA and RNA polymerases immobilized in vivo in large transcription factories (Iborra et al., 1996). DNA polymerase covalently linked to a surface via amide-bond formation retained 10% of its solution-phase activity when its active site was masked by substrate during immobilization (Hwang et al., 2002; Hwang et al., 2004). In contrast, Taq DNA polymerase fused to a human serum albumin (HSA) binding protein yielded little extension product after affinity-immobilization to a HSA-coated surface (Nilsson et al., 1997). Transcriptional fidelity, reliability and yield is increased by covalently linking the polymerase domain to a sequence non-specific DNA binding protein to impart a sliding grip on the minor groove (Pelletier, 2000; Wang et al., 2004). This enhanced polymerase processivity 2-fold relative to Taq and 12-fold relative to other high-fidelity polymerases, yielding a transcription rate of 3.8 kilobasepairs (kb) per minute. This rate correlates well with replication-fork movement of 0.5 to 5 kb per minute in eukaryotic chromosomes, but is well below prokaryotic rates of almost 100 kb per minute (Kornberg & Baker, 1992). Immobilizing polymerase to perform PCR amplification could be readily integrated into Lab-on-a-Chip devices (Hwang et al., 2003).
SPR-Induced Heating of Au Surfaces: Titanium:sapphire (Ti:sapphire) laser pumping is the method most frequently used to dissipate heat from Ag and Au nanoparticles (Boyer et al., 2002; Halte, et al., 1999; Hu & Hartland, 2002) and Au—Si nanoshells (Hu et al, 2003) and to inactivate Au-surface-associated proteins (Huettmann, et al., 2003; Radt et al, 2001). However, heating for thermal melting, binding, or desorption of DNA bases and nucleotides interacting with Au surfaces has been induced only by changing bulk solution temperature (Demers et al., 2002; Jin et al., 2003) or by inductive coupling (Hamad-Schifferil et al., 2002). Inductive coupling of radio-frequency magnetic field to 1.4-nm Au nocrystals with a power output of 1-4 watts increased apparent temperature 13° C., but this increase is insufficient for PCR thermal cycling.
Femtosecond laser pulses excite nonlocalized Au electrons to create a highly nonisothermal electron distribution that relaxes within 500 fs by electron-electron scattering to establish a new distribution corresponding to a higher temperature (Link & El-Sayed, 2003). Temperature (T) increased in NP with diameters from 5 to 50 nm in proportion to pump laser flux (intensity, Io/spot size, σ), a function of sample absorbance (A), and the inverse of sample heat capacity, Cp, Au concentration, and cell pathlength (l), respectively, according to Beers Law:
                              Δ          ⁢                                          ⁢          T                =                                                                              I                  o                                ⁡                                  (                                      1                    -                    ζ                                    )                                            2                        ⁢                          (                              1                -                                  10                                      -                    A                                                              )                                                          σ              ⁡                              [                Au                ]                                      ⁢                          C              p                        ⁢            l                                              (        1        )            
In Eq 1, (1−ζ)2 with ζ˜0.04 accounts for pump laser scattering in the sample cell. Temperature increases (ΔT) of 40 K were produced in 15 nm Au NP using 0.2 μJ/pulse/6×10−4 cm2 pump power from a regeneratively amplified Ti:sapphire laser (λ=780 nm; 0.5 mJ/pulse/; 120 fs fwhm sech2 deconvolution; 1 kHz repetition rate) (Hu and Hartland, 2002). Much higher laser fluxes were required to inactivate Au-NP-bound proteins. Thirty-five ps pulses at 527 nm with 50 mJ/cm2, and 16 ns pulses at 450 mJ/cm2 inactivated 80% and 100% of alkaline phosphatase bound to 15-nm NP, respectively (Radt et al, 2001). These studies did not report rapid cycling of SPR-induced thermal dissipation to anneal, elongate or denature DNA strands but they are useful to design conditions for SPR-based PCR. Ti:sapphire laser pumping, continuous Argon-ion lasing, or alternate optical sources that stimulate surface plasmon resonance can achieve ΔT=17 K and ΔT=39 K required for elongation and denaturation, respectively, relative to an ambient temperature of 55° C. without inactivating Taq or Phusion™ polymerase.
Characteristic Time Scale for Temperature Dissipation: Gold electrons excited by femtosecond laser light exchange energy with the environment via pump-power dependent electron-phonon coupling (Link & El-Sayed, 2003). Particle temperatures in aqueous Au sols dissipate with time, T(t), relative to the surrounding (Ts) and initial (Ti) temperatures, according to a phenomenological stretched exponential function (Hu & Hartland, 2002):
                                                        T              ⁡                              (                t                )                                      -                          T              s                                                          T              i                        -                          T              s                                      =                  exp          ⁢                      {                          -                                                (                                      t                    /                    τ                                    )                                β                                      }                                              (        2        )            
where τ=0.64 picoseconds nm−2×R2 for R in nm, and β=0.6 and 0.7 for 2R=5 nm to 15 nm and 26 nm to 50 nm particles, respectively. Observed characteristic decay times increased from 10 ps for 2R=5-nm Au NP to 380 ps for 2R=50-nm Au NP. The energy-dissipation time-scale, τd, may be estimated in general (Wilson et al, 2002) by equating the heat capacity of radius R particles, 4/3πR3 Cp, to the heat capacity of adjoining thermal diffusion layer of thickness ld=(ατd)1/2, where α is thermal diffusivity. The heat capacity of the diffusion layer is 4πR2ldCf. The time scale is then:
                              τ          d                =                                            r              2                        ⁢                          C              p              2                        ⁢                          ρ              f                                            9            ⁢                          C              f                        ⁢                          k              f                                                          (        3        )            
Eq. 3 shows τd increases proportional to particle surface area for 2R=5 nm or larger and inversely with thermal conductivity, kf, and heat capacity, Cf, of surrounding medium, which is consistent with an absence of interface effects. Immobilizing alkanethiol-capped DNA onto Au NP changes the interface thermal conductance, G, which has units of Wm−2K−1, and delays energy exchange between surface Au atoms and surrounding molecules. The characteristic decay may then be estimated as (Wilson et al, 2002):
                              τ          i                =                              rC            p                                3            ⁢            G                                              (        4        )            
Although G>20 MW m−2K−1 was reported for dodecanethiol-terminated 2R=3-5 nm Au NP, a potential electron effect at the Au-alkanethiol interface increased the observed decay about 10-fold relative to the value obtained with Eq 4. Amplification of 110- and 536-bp fragments of human β-globin gene would take about 66 and 330 milliseconds, respectively at a rate of 1667 bp per sec (Kornberg & Baker, 1991).
Characteristic Length Scale for Temperature Dissipation: Using a A20 spacer (Jin et al., 2003) between the forward/reverse primer pairs and the Au surface gives total lengths (fragment plus spacer) of 44 and 189 nm for 110- and 536-bp fragments of human β-globin, respectively, since 106 nucleotides occupy a linear distance of 3.4×105 nm (Molecular Biology at NCBI, 2004).
Commercially-Available Thermal Cyclers vs. Real-Time PCR: PCR steps in conventional block thermal cyclers take 20 to 60 seconds each. Hours are required for 35-45 cycles to achieve end-point gene levels that are detectable by subsequent ethidium bromide or fluorescent staining of gels. Real-time PCR methods that quantify genes with fluorescent labels are more reproducible, have a wider dynamic range and eliminate risk of carryover contamination (Vet & Marras, 2005). Real-time fluorescent-label methods avoid second round amplification, false positives, or confirmational sequencing. Fluorescent SYBR green dye intercalates dsDNA during annealing and extension steps, but melt temperatures, Tm, must be analyzed to distinguish signal from SYBR interaction with desired amplicons from non-specific interaction with primer dimers. Using FRET from donor 3′-fluorescein to adjacent acceptor cyanine dye Cy5 or 5′-LC Red 640 labeled on amplicon-specific hybridization probes that are 23-35 bp long reduces non-specific background signal (Wittwer et al., 1997). LightCycling™, the fastest real-time PCR method, heats template samples in glass capillaries by forced air and continuously monitors DNA formation by fluorescence. LightCycling™ can detect ˜100 initial copies and finish in 10 to 15 minutes.
SPR decreases the intensity of light reflected at a specific angle from a conducting gold film adjacent to a dielectric medium (glass and sample). The setup for SPR with planar- or NP-Au surfaces is shown in FIG. 1. At the angle of minimum reflectance, incident light photons excite delocalized metal electrons, causing them to oscillate collectively and dissipate light energy. The minimum angle varies with the sample refractive index close to the glass surface. β-globin template that binds to immobilized forward/reverse primers increases the local refractive index (RI), shifting the angle of minimum reflectance in proportion to mass concentration, producing a SPR sensorgram. RI changes are detectable within ˜200 nm of flat Au surfaces and within 10 to 20 nm of 40-nm Au NP